The garlic extract‐loaded nanoemulsion: Study of physicochemical, rheological, and antimicrobial properties and its application in mayonnaise

Abstract In this research, garlic extract (GE)‐loaded water‐in‐oil nanoemulsion was used as a novel preservative and antioxidant in mayonnaise. GE (5%, 10%, 15%, and 25%) as a dispersed phase, olive oil as a continuous phase, and polyglycerol polyricinoleate (PGPR) as a low HLB surfactant, with a constant surfactant/garlic extract ratio (1:1), were used in the formulations of water‐in‐oil nanoemulsions. The properties of the active nanoemulsion, including droplet size, free radical scavenging capacity, antimicrobial activity against gram‐positive (Staphylococcus aureus [25923 ATCC]), and gram‐negative (Escherichia coli H7 O157 [700728 ATCC]) were evaluated. The results showed that the mean droplet size of nanoemulsion increased from 62 to 302 nm and antioxidant capacity was also improved from 95.43% to 98.25% by increasing GE level from 5% to 25%. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) showed that antimicrobial activity against S. aureus could be observed only in high levels of GE (25%) in the formulation of nanoemulsion. The results of the total count analysis showed that the GE‐loaded nanoemulsion (NEGE) was effective against the microorganisms, particularly after 4 months of storage. The incorporation of GE and NEGE did not affect significantly the acidity of different mayonnaise samples; however, they affected the concentration of the primary product of lipid oxidation. Adding GE and NGE did not significantly affect the rheological properties of mayonnaise and all samples showed shear‐thinning behavior. Sensory evaluation showed that the samples with NEGE had higher scores in texture, spreadability, and mouthfeel, while the control samples had better scores in appearance, color, taste, and total acceptance. In general, the samples containing free GE (not encapsulated) had the lowest scores in all organoleptic characteristics.


| INTRODUC TI ON
Mayonnaise is oil-in-water emulsion salad dressing and it is widely consumed around the world. Mayonnaise is usually prepared from oil that is dispersed in an aqueous phase and consists of egg or egg yolk, vinegar, sugar, salt, spices (especially mustard), and some food additives, including stabilizers and preservatives. As there is no sufficient heat treatment in the mayonnaise preparation, preservatives are usually used in the formulation (Da Silva & Franco, 2012). The salts of organic acids, such as potassium sorbate (PS) and sodium benzoate (SB) are the most common preservatives that are used in mayonnaise formulation because of their antifungal and antimicrobial activities. However, some health adverse effects of synthetic preservatives have been reported by different researchers. Afshar et al. (2013) showed that 560 mg kg −1 of SB had reduced weight and crown-rump length of the fetus of mice. Sohrabi et al. In recent years, due to possible risks and adverse effects of synthetic preservatives, using natural preservatives such as herbal extracts has received a great deal of attention. Furthermore, as mayonnaise is composed of 70%-80% oil, lipid oxidation is another reason for the quality deterioration in mayonnaise. Herbal extracts can have a high potential in reducing lipid oxidation due to their high content of phenolic compounds. Garlic contains a variety of biologically active compounds, including sulfur compounds, saponins, phenolic compounds, and polysaccharides. The main active components of garlic include its sulfur compounds, such as di-allyl thiosulfonate (allicin), diallyl sulfide, diallyl disulfide, and diallyl trisulfide. Numerous studies have shown that garlic and its bioactive ingredients possess antioxidant, anti-inflammatory, antibacterial, antifungal, immune system regulator, cardiovascular protection, anticancer, liver protection, gastrointestinal improvement, antidiabetes, anti-obesity, protection of the body's nervous system, and kidneys protective properties (Shang et al., 2019).
Garlic extract has limitations for direct use in food products due to its high volatility and its effects on organoleptic properties. Therefore, it is better to encapsulate it and then incorporate it in food formulations. In recent years, many delivery systems including lipid-based carriers and biopolymer-based systems have been developed to encapsulate bioactive compounds in the food industry. One of the most common lipid-based encapsulation systems is nanoemulsions that are prepared differently such as high-and lowenergy methods (Hassanzadeh et al., 2018).
Nanoemulsions are fine droplet emulsions that have a droplet size below ~200 nm and show relatively higher kinetic stability against flocculation, coalescence, and creaming than macroemulsions (conventional emulsion) due to their smaller droplet size (McClements, 2011;Saberi et al., 2013;Salvia-Trujillo et al., 2015). Fabrication of nanoemulsions for encapsulation and controlled delivery of bioactive ingredients is one of the functional areas of nanotechnology in the food industry (Habibvand et al., 2022;Velikov & Pelan, 2008;Yang et al., 2012). Rabbani et al. (2021)  Bactris gasipaes fruits. Carotenoids from B. gasipaes fruits were prepared by ultrasound using sunflower oil to prepare an oil-in-water food emulsion similar to typical mayonnaise with a reduced fat percent. Their results present a pleasant idea for carotenoid incorporation into new foodstuffs, representing a new model to design functional products with more bioavailable lipophile bioactive components. This design can be applied to improve the functionality of food systems with insufficient nutritional properties. Therefore, the aims of the present study were to produce the GE-loaded water-inoil nanoemulsion (NEGE) and then using of NEGE in mayonnaises as a preservative, antioxidant, and functional nutritional agents.

| Preparation of garlic extract
First, the garlic cloves were soaked in water for better separation of the skin, and then the skin and bottom of the garlic cloves were removed and the garlic cloves were washed. After washing, the garlic was poured into a mixer, and after crushing it completely, it is mixed with distilled water at a ratio of 1:1. The mixture was then stored at a temperature below 15°C for 24-48 h. Finally, the mixture was passed through a cloth strainer to separate the coarse garlic particles and the filtered liquid was passed through a filter paper.
Finally, the extract was centrifuged at a rotational speed of 4248 g for 15 min, and thus the garlic extract was obtained.

| Preparation of nanoemulsions
After preparing the garlic extract, the olive oil was mixed with the PGPR surfactant at a ratio of 1:1 with dispersed phase (GE) while stirring. Garlic extract was then added dropwise and slowly to the stirring oil and surfactant mixture at a rotational speed of 29.5 g.
The prepared mixture was continued to mix at a rotational speed of 57.82 g for 20 min and transferred to an ultrasonic homogenizer for further homogenization and the desired nanoemulsions were prepared (Mohammed et al., 2021).

| Droplet size distribution analysis
To measure particles, all of the nanoemulsions are diluted at a ratio of 1:50 in oil to prevent multiple particle scattering. The mean particle size (Z average ) and polydispersity index (PDI) were determined by dynamic light scattering (Malvern Instrument) at the temperature of 25°C and an angle of 90°.

| Free radical scavenging capacity
The antioxidant properties of the prepared nanoemulsions and garlic extract were measured using DPPH method. In this method, 4 mL of 60 μM free radical methanol solution of DPPH was mixed with 0.2 mL of the prepared nanoemulsion and kept at room temperature for 60 min. Then, the absorbance of the solution was read at 517 nm using a spectrophotometer. Also, 4 mL of DPPH with a sample containing 0.2 mL of methanol was determined as a control sample, and radical scavenging capacity was calculated using the relevant below formula (Equation 1) (Hasanzadeh et al., 2017). and sterile saline. TSB culture medium was prepared separately and 100 μL of it was poured into 7-well plates. TBS culture was separately prepared and various dilutions of nanoemulsions in this medium in a 96-well plate were prepared. Firstly, 100 μL of nanoemulsion with 100 μL TBS culture were mixed and after stirring, 100 μL of this mixture was transferred to the next wells and diluted by 100 μL of TBS culture again, and this was repeated five times. And so, six different dilutions of nanoemulsion were prepared in 6 wells of the plate, and the seventh was considered as the control without the nanoemulsion. 1/2-1/64 dilutions of the nanoemulsions were formed. Next, 20 μL of prepared dilution of bacteria were inoculated in 6 wells containing culture medium and nanoemulsions. In the seventh well, only 100 μL of culture medium was added and considered a control.

| The minimum inhibitory concentration and minimum bactericidal concentration
The prepared dilution of bacteria was equal to 0.5 McFarland (approximately contain 1.5 × 10 8 CFU mL −1 ), but to obtain more accurate results, 0.5 McFarland was diluted by sterile saline to achieve approximately 5 × 10 5 CFU mL −1 . Finally, 96-well plates were placed at 37°C for 18 h and then MIC was determined by comparing the turbidity of treated wells with control wells. To determine the MBC, from bacteria treated 96-well plate, 1 dilution lower than MIC dilution and 2 dilutions higher than it, were cultured linearly on Mueller-Hinton agar medium. The lowest concentration, at which the line growth was not found on agar, was considered MBC (Andrews, 2001).

| Preparation of mayonnaise
The mayonnaise samples were prepared according to the basic formulation of low-fat mayonnaise which is now produced commercially. The control mayonnaise recipe contained the following ingredients based on the percentage (w/w): vegetable oil 40, egg yolk 9, and vinegar 9 (11% w/v acetic acid), salt 1, sugar 5, mustard 0.3, stabilizer 0.15, modified starch 2, and water 42.55. The mayonnaise samples were prepared using nanoemulsion as disperse phase or garlic extract as the aqueous phase. Also, the amounts of preservatives including SB and PS varied ranged from 0, 375, and 750 ppm according to the experimental design. The mixtures composition in the mayonnaise formulation is shown in Table 1. For mayonnaise preparation, a standard mixer was used (Moulinex, HM312). First, the modified starch and stabilizers were dissolved in an amount of oil separately. Then dissolved starch and dry matters were mixed followed by the addition of the dissolved stabilizers and about onethird of water. Then a small amount of the oil was added. The ingredients were blended for approximately for 5 min. Afterward, one-third of vinegar was slowly added to the mixer. Finally, the remaining oil, water, and vinegar were gradually added and admixed in a mixer. The prepared mayonnaise was transferred to glass bottles and stored at room temperature (25°C) for further analysis. international standards. These assessments were done monthly throughout the 4 months of storage at 25°C.

| Acidity measurement
About 15 g of the prepared mayonnaise was mixed with 200 mL of distilled water, then 3-4 drops of phenolphthalein was added and titrated with NaOH 0.1 N until the first color changed to pink, and finally the acidity value calculated by the corresponding formula (Tavakoli et al., 2021).

| Oxidation stability (peroxide value)
IDF standard method was used for determining the peroxide value of all samples. To determine the peroxide value, 8.9 mL methanolchloroform (3 + 7) (V/V) was mixed in a glass test tube using a mixer for 2-4 s and then 50 μL of ammonium thiocyanate solution was added and then samples were taken for 2-4 s and mixed with a vortex mixer. Finally, 50 μL ferrous sulfate (II) was added and the samples were mixed for 2-4 s with a vortex mixer.
After 5 min of incubation at room temperature, absorbance versus blank (all indicators except sample) were read by spectrophotometer, and then peroxide value was calculated based on the following equation (Equation 2). The whole procedure is performed in the dark for 10 min.
where A s = sample absorbance; A b = Blank absorbance; m = slope, obtained from the calibration curve, m 0 was the sample weight and 55.84 was the atomic weight of iron.

| Steady and oscillatory shear rheological analysis
Flow behavior and oscillatory tests were performed using a rheometer (Anton Paar Physica MCR300). Flow properties of the mayonnaise samples were determined at 20°C using a parallel stainless steel plate with 20 mm diameter in the shear rate range of 0.1-100 s −1 . To determine the flow behavior, the experimental data were fitted to a Power-law equation (Equation 3): where τ is the shear stress (Pa), is the shear rate (1/s), K is the consistency index (Pa.sn), and n is the flow behavior index.
The dynamic oscillatory tests were conducted over a frequency range of 0.1-50 Hz at a constant strain of 0.5% (within the linear viscoelasticity range that was previously established by the strain sweep tests). Data were collected and rheological parameters were calculated using a rheometer software program. Storage modulus (G′) and loss modulus (G″) and tan (delta) versus frequency was measured for all the samples.

| Sensory evaluation
After 1-day of storage at room temperature, the organoleptic tests were done for the mayonnaise samples. The mixed 5-point hedonic scale was used (scale, 1 = the least acceptable; 5 = the most acceptable). The texture, appearance, odor, taste, and overall acceptance were measured by 12 panelists. Some bread and a cup of water were provided between samples to clean the palate.

| Statistical analysis
In the first stage of this study (fabrication of nanoemulsions), experiments were performed in duplicate at least for calculating the mean and standard deviation. One-way ANOVA and Tukey tests were used for the analysis of data at (α = 0.05) using Minitab software (version 17) for mean treatments comparison. D-optimal design (preservative level in tree level and types of enriched mayonnaises) was used by Design-Expert software (version 10) for analyzing mayonnaises in the second stage. Table 1 shows mayonnaise formulated with nanoemulsion, garlic extract, and preservatives.

| Droplet size analysis
The results of DL for the GE-loaded water-in-oil nanoemulsions showed that the particle size was increased significantly with increasing volume fraction (garlic extract). As shown in Table 2, by increasing the percentage of GE from 5% to 25%, the particle size has increased from 62 to 302 nm. This indicates that the particle size of water-in-oil nanoemulsions is affected by the volume fraction. in water-in-oil nanoemulsion formulations from 10% to 40%, their particle size increased from 13 to 186 nm. Also, Porras et al. (2005) studied the production of water-in-oil nanoemulsions and reported that the particle size was increased from 30 to 120 nm by increasing the aqueous phase ratio from 5% to 25%. Also, the average droplet size between 132 ± 2.0 and 145 ± 1.0 nm was obtained in O/W pepper nanoemulsions produced by high-pressure homogenization with an inverse ratio between particle size and dispersed phase ratio (Galvão et al., 2018).

| Free radical scavenging capacity
The results obtained from the DPPH method for water-in-oil nanoemulsions showed that in general, this type of nanoemulsion has more free radical scavenging capacity than oil-in-water nanoemulsions. This can be due to the fact that there is some phenolic compounds in the aqueous phase of water-oil nanoemulsions and the antioxidant ability of the oil itself (Hassanzadeh et al., 2018).
As can be seen in Figure 1, there is a significant differences be-

| MIC and MBC of nanoemulsions
Although to our best knowledge limited research has been pub-  Table 3).
By comparing the antimicrobial activity of oil-in-water nanoemulsions (Hassanzadeh et al., 2018) and water-in-oil nanoemulsions, it can be observed that water-in-oil nanoemulsions have lower antimicrobial activity. This could be due to the higher intensity of volatiles in garlic essential oil than in its extract. In a similar pattern with oil-in-water nanoemulsions, it was found that they have more antimicrobial activity against gram-positive bacteria than gram-negative bacteria (Hassanzadeh et al., 2022). cell protein synthesis is one of the proposed mechanisms (Feldberg et al., 1988). It is stated that a major part of the antimicrobial properties of garlic is related to allicin and its metabolites. These compounds exert their antimicrobial activity through specific inhibition of the enzyme acetyl-coenzyme A-synthetase. Inhibition of this enzyme inhibits the biosynthesis of lipids and fatty acids and ultimately impairs cell viability.
One of the characteristic properties of garlic organosulfur compounds is their ability to penetrate through membrane phospholipids. Among the compounds in aqueous garlic extract, diallyl thiosulfate is the most effective compound in the biological and antimicrobial activities of garlic. As shown in Table 3, due to the encapsulation of most of the volatile and functional compounds of TA B L E 2 Particle size obtained from DLS for water-in-oil nanoemulsions.

| Microbial characteristics
The results of the total count showed that the studied factors have been effective against a number of microorganisms, particularly after 4 months of storage. SB, as a preservative, has played the most important role in reducing total count. The addition of GE as well as nanoemulsions containing GE also reduced the total count ( Figure 2). As discussed in the previous part, GE can reduce the microbial load of mayonnaise due to its organosulfur compounds such as allicin and similar compounds. However, the nanoemulsions containing GE may be due to the slow and continuous release of volatile compounds that shows higher effects on the total count.   Their results revealed that Ant-eCsNe holds good potential to be applied as a food preservative to reduce fungal and aflatoxin contamination causing deterioration of stored maize.

| Acidity measurement
Acidity is one of the most important properties of mayonnaise, dressings, and sauces for recognizing the growth and survival of pathogenic bacteria (Tavakoli et al., 2021). Acetic acid is the main acid in mayonnaise, which presents as various kinds of vinegar. The effects of acidity on microorganisms are as follows: (1) the impact of pH alone, (2) the effect of undissociated forms of a special acid, and (3) the particular effects of organic acids (Jalilzadeh et al., 2018). As shown in Figure 3, statistical analysis of data demonstrated that there is no significant difference between the acidity of the differently formulated mayonnaises. This means that the addition of GE and nanoemulsions containing GE did not affect the acidity (p > 0.05). In contrast, the addition of SB significantly affected the acidity of mayonnaises and decreased from 1.15 to 0.85 by changing SB from 0 to 750 ppm (p < 0.05). Similar results were recorded by Tavakoli et al. (2021) as they reported the higher pH value in mayonnaise samples containing preservatives compared with the control sample during storage. Gorjian et al. (2021) compared the effect of nanoliposome and nanoniosome containing myrtle extract as a natural preservative and SB on microbial, physicochemical, and organoleptic characteristics of mayonnaise and concluded that the highest pH (4.2) was seen in sauce sample containing SB.

| Peroxide value
Peroxide index is a major criterion for measuring hydroperoxides which are the primary products of lipid oxidation. Hydroperoxides were increased and then decreased during oxidation, and this process continues due to their successive formation and degradation [19]. Since mayonnaise is an oil-in-water emulsion and its oily phase which is in contact with a large area of water, it is very prone to oxidative damage. Therefore, an increasing trend in the number of peroxides due to the intensification of oxidation can be observed for sauce samples in particular during long storages. As can be seen in Figure

| Rheological analysis
Steady-state shear rheology The power-law model is widely used to describe the relationship between shear stress and shear rate in many oil-based food products and emulsions (Rahbari et al., 2015). The shear stress data-shear rate data of different mayonnaise samples were fitted in power-law model with high coefficient of determination (R 2 ). Table 4 shows the values of the power-law model parameters for each of the mayonnaise samples. In general, the samples containing nanoemulsion showed the lowest flow behavior index value but the highest consistency index value. The role of water-in-oil nanoemulsion in increasing the dispersed phase content of mayonnaise (as oil-in-water nanoemulsion) can be considered as the reason for this change in flow behavior.

Dynamic (oscillatory) rheological behavior
In the frequency sweep test, a gradual increase in storage modulus and loss modulus of treatments was observed with increasing frequency (Figure 6).  The maximum scores for oscillatory measurements of mayonnaise samples were related to mayonnaise formulated with WPI, while the least score was shown by mayonnaise formulated with SPI.

| Sensory evaluation
Results from organoleptic characteristics (appearance, color, taste, texture, spreadability, mouthfeel, and total acceptance) that were evaluated by panelists showed that samples containing nanoemulsions of GE had higher scores in texture, spreadability, and mouthfeel while the control samples had better scores in appearance, color, taste, and total acceptance. In general, samples containing GE

ACK N OWLED G M ENTS
Ilam University is appreciated because this research is financially supported by the research department of Ilam University with a Grant ID of 04-IRILU-Vt-000061-21.

FU N D I N G I N FO R M ATI O N
The author(s) received no financial support for the research, authorship, and/or publication of this article.

CO N FLI C T O F I NTE R E S T S TATE M E NT
We wish to confirm that there are no known conflicts of interest associated with this publication.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author, upon reasonable request.